Fantastic ribonucleoprotein complexes and how to study them. Architectural principles of regulatory assemblies in bacteria.
University of Cambridge
Doctor of Philosophy (PhD)
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Dendooven, T. (2020). Fantastic ribonucleoprotein complexes and how to study them. Architectural principles of regulatory assemblies in bacteria. (Doctoral thesis). https://doi.org/10.17863/CAM.63172
The fate of a bacterial RNA transcript is controlled by three key players: ribonucleases, RNA binding proteins (RBPs) and small regulatory RNAs (sRNAs). Together these form an information-rich regulatory network that exercises post-transcriptional control of gene expression. A key RBP in bacteria is Hfq, which is best studied as a facilitator for sRNA-mRNA pairing. In Pseudomonas aeruginosa, however, Hfq is known to modulate translation of more than 100 genes in cooperation with an effector protein, Crc, by sequestering the ribosome binding site of the corresponding mRNA molecules. In this thesis, Cryo-EM studies on target RNAs such as amiE and rbsB, reveal how Hfq presents multiple A-rich motifs in the mRNA 5ʹ-end to Crc and triggers assembly of higher order complexes. The structures suggest that secondary structure elements and the sequence of the target RNA determine the quaternary structure of the Hfq-Crc translation-repression assembly, introducing a degree of structural diversity that is accommodated by polymorphic interaction surfaces on both Hfq and Crc. In Escherichia coli, the homolog of the same RBP (Hfq) can modulate sRNA lifetime by repurposing the conserved ribonuclease PNPase (polynucleotide phosphorylase) into an RNA chaperone. Cryo-EM structures presented here show how the flexible KH-S1 portal of PNPase generally guides the 3ʹ-end of the sRNA sponge 3ʹETSleuZ to the catalytic core for digestion but reroutes the sRNA when bound to Hfq. In particular, an A-rich motif in the 5ʹ-end of 3ʹETSleuZ is presented to the KH and S1 domains by the Hfq distal side, while the 3ʹ-end is sequestered on the proximal side. Gel shift- and activity assays suggest that these repurposed ‘RNA carrier’ complexes protect sRNAs from other ribonucleases such as RNase E and RNase III, and potentially lend their KH-S1 portal as an interaction platform to promote sRNA/RNA target pairing. A fraction of PNPase in the cell associates with the endoribonuclease RNase E to form the E. coli ‘RNA degradosome’, a multi-enzyme ribonuclease assembly responsible for the bulk RNA turnover in the cell. Other partner enzymes include the DEAD-box helicase RhlB and Enolase. The core of the assembly, RNase E, has a long C-terminal scaffold domain that has a conserved natively unstructured character. The subcellular localization of the RNA degradosome, anchored to the cell membrane, provides a spatial component to post-transcriptional gene regulation. In this work, holistic experimental strategies to study this molecular machine are developed. Reproducible reconstitution of a truncated version of the RNA degradosome was achieved with a mix of detergents. Using small angle X-ray solution scattering (SAXS) combined with conformational ensemble computation indicates that the truncated degradosome is a highly flexible system with extended scaffold domains but can adopt more compact conformations when 9S rRNA is bound. Complementary cryo-EM studies of the catalytic core and subsequent 3D variability analyses reveal three modes of molecular motion of the RNase E protomers, adding to the overall flexibility of the RNA degradosome. Lastly, cryo-electron tomography studies of the truncated degradosome tethered to lipid vesicles provide the first visualization of the degradosome in its native environment and hint towards a degree of structural order in its components. Subsequent sub-tomogram averaging approaches to investigate putative membrane-bound RNA surveillance complexes set a foundation for future structural studies.
Cryo EM, Cryo ET, Post-transcriptional gene regulation, Small regulatory RNA, sRNA, P. aeruginosa, E. coli, RNA degradosome, PNPase, Hfq, Crc, Bacteria
This record's DOI: https://doi.org/10.17863/CAM.63172
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